The proposed research will investigate how protease inhibitors (e.g. PMSF, TLCK, TPCK) and protease substrates (e.g., TAME, BAEE, ATEE, TME) inhibit the binding of steroid hormones to cellular receptors and serum proteins. We will study the effects of these compounds on the binding of deoxy-corticosterone (DOC) to its receptor in the dog kidney (MDCK) cell line, estrogen to alpha-fetoprotein, and glucocorticoids, mineralocorticoids, and sex hormones their receptors in rat tissues. Protease substrates containing D-amino acids and/or hydrolysis products such as tosylarginine will be used to determine if inhibition of steroid binding to receptors is by action at the site which binds the steroid (competive inhibition) or if inhibition occurs by an allosteric mechanism (noncompetive inhibition). Nonradioactive and radioactive PMSF, TLCK, and TPCK will be used to determine the structure of their binding site in alpha-fetoprotein. This structure will be compared with that in serine residue proteases to determine what similarities in mechanisms exist between an estrogen binding protein and proteases. Analogues of steroid hormones which contain reactive chloroketone groups at different sites in the steroid will be synthesized and tested for their ability to inhibit steroid hormone binding to receptors. Our results with protease substrates will be applied to purifying steroid receptors by affinity columns containing a protease substrate couple to an agarose matrix. We expect that our studies will be useful in developing techniques for regulating the binding of steroid hormones to their receptors. This should find application in such diverse areas as reproductive biology, control of malignant cell growth, hyperaldosteronism, and arthritis.